Circadian rhythms control the timing of numerous physiological processes over a 24-hour period, including sleep-wake cycles, thermoregulation, feeding, metabolic regulation and hormone production. At the heart of the molecular network that constitutes the circadian clock are the core transcription factors CLOCK and BMAL1 that heterodimerize, and in conjunction with the transcriptional machinery, direct rhythmic expression of clock-controlled genes (CCGs). A critical component of circadian gene expression is linked to CLOCK, a known histone acetyltransferase, which directs rhythmic acetylation of histone H3 lysine 9/14 as well as BMAL1. In terms of circadian transcription, little is known about the transcriptional machinery that works in concert with CLOCK and BMAL1 to direct circadian gene expression. The goal of this proposal is to elucidate the mechanisms of regulation of the mammalian sirtuins, class III histone deacetylases (HDACs), on circadian gene expression and biological rhythmicity in vivo. SIRT6 is a known chromatin-associated HDAC that directs deacetylation of histone H3 lysine 9 at relevant gene promoters, but SIRT6 has not been implicated in circadian biology. Of particular importance, SIRT1 is responsible for deacetylation and regulation of BMAL1, as well as deacetylation of H3 lysine 9/14 at circadian gene promoters. Surprisingly, SIRT1 is a nuclear sirtuin that is mostly localized in the nucleoplasm, therefore it in unclear whether the efficiency of SIRT1 is suited for histone targets or better directed towards non-histone proteins such as BMAL1. It is hypothesized that a functional interplay exists between SIRT6 and SIRT1 in modulating circadian gene expression, and that the subcellular localization of these sirtuins may dictate efficiency of deacetylase activity towards histone versus non-histone targets. It is hypothesized that the HDAC SIRT6 is associated with the core circadian transcription factors, CLOCK and BMAL1, and may be involved in modulating circadian gene expression by deacetylating H3K9 at circadian gene promoters, resulting in transcriptional repression and subsequent oscillation of CCG expression. To test this hypothesis, the use of wild-type and SIRT6 knockout mice as well as mouse embryo fibroblasts (MEFs) will be utilized to determine SIRT6-mediated effects on circadian gene expression in vitro and in vivo, and how these actions differ from SIRT1.